Substituted phenyl boronic acid containing polymers and methods of use

ABSTRACT

The disclosure relates to cationic polymers functionalized with substituted phenylboronic acid groups and to methods of using the same to treat metabolic and gastrointestinal disorders.

RELATED APPLICATIONS

This application filed under 35 U.S.C. 111(a) is a continuation ofInternational Application No. PCT/US2020/051506, filed on Sep. 18, 2020,which claims the benefit of U.S. Provisional Application No. 62/903,328,filed on Sep. 20, 2019. The entire teachings of the above applicationsare incorporated herein by reference.

BACKGROUND OF THE INVENTION

Type-2 diabetes mellitus (T2DM) affects about 11.3% of the U.S. adultpopulation, with 35% of the U.S. adults having pre-diabetic symptoms.U.S. healthcare costs due to diabetes are approaching $200 billionannually. The incidence of T2DM continues to increase in parallel withthe obesity epidemic, and a major portion of present treatment for T2DMconsists of a regimen of oral medications that may be suboptimal formany subjects, in part because of side effects associated with systemicabsorption of their medication. Bariatric surgery to bypass or excludethe duodenum from the digestive tract has been shown to improve T2DM.The Diabetes Surgery Summit recommends bariatric surgery to treat T2DMin some obese patients (grade III obesity), and that bariatric surgeryshould be considered for treatment of other patients. (See, e.g.,Koliaki, C. et al., BMC Endocr Disord. (2017) 17:50 DOI10.1186/s12902-017-0202-6.)

Analysis of the typical diabetic patient's path from first line drugs toinsulin and on to surgery and other highly invasive treatments revealsstriking gaps, not limited to ineffective treatments and clinicalinertia. Surgery and other solutions also have failed to achievewidespread adoption. The addition of specialist clinicians in the carepathway has contributed to those failures. Accordingly, an effectivetreatment in the hands of the primary care physician would likely reacha much larger segment of the subject population than those which requirea specialist, such as an endocrinologist, a gastroenterologist, or asurgeon, and would therefore have much greater impact.

US 2006/0134062 disclose polymers in which certain arylboronic acidmoieties are bonded to the polymer backbone through long linkers, andthe use of such polymers as inhibitors of lipase. The polymer backboneis said to be not critical for lipase inhibition.

U.S. Pat. No. 7,943,713 disclose certain polyamine boronic acidderivatives and their use to increase paper wet web strength and wetstrength. The preferred polymers are characterized by aryl boronic acidmoieties that are directly bonded to a carbon atom in the polymerbackbone, or bonded to a carbon atom in the polymer backbone through anamide linkage.

WO 2017/024237 discloses certain cationic polymers and use of thepolymers for complexing mucus to form an occlusive barrier in theduodenum. Seno, M. et. al, Materials Science and Engineering C, 62(2016) 474-479, discloses certain pH- and sugar-sensitive multilayerfilms that are composed of phenylboronic acid-modified poly(allylaminehydrochloride) and pol(vinyl alcohol). Sato, K. et al, Langmuir, 2014,30, 9247-9250, also discloses multilayer films that are composed ofphenylboronic acid-modified poly(allylamine hydrochloride) and pol(vinylalcohol).

A need therefore exists for new medications and non-invasive methods fortreating subjects with T2MD and related metabolic disorders.

SUMMARY OF THE INVENTION

The invention provides polymer compositions for forming a physicalbarrier in the gastrointestinal (GI) tract of a subject between theintestinal lining and the luminal contents. The polymers of theinvention are mucin-interacting agents which form a physical barrierin-situ by interaction with resident mucin in the GI tract.

The inventors have discovered that the incorporation of pendantsubstituted phenyl boronic acid moieties into certain cationic polymersdramatically improved the mucin-complexing activity of the polymers.

As shown in the examples, the polymers described herein have improvedmucin and mucus complexing activity in comparison to comparable cationicpolymers, and can effectively condense mucin and mucus at the pH of theduodenum. The polymers bind tightly to mucus at the pH of the duodenumand once bound to mucus are resistant to removal by high concentrationsof salts (e.g., 1M NaCl). The resulting polymer-mucus complexes havedramatically different properties in comparison to free mucus.

This disclosure relates to cationic polymers that contain pendantsubstituted phenyl boronic acid groups, which are bonded directly orindirectly to the polymer backbone through an amine or amide bond, andto a method of treating metabolic diseases that include administering atherapeutically effective amount of such a polymer to a subject in needthereof.

Another aspect of the invention is a pharmaceutical compositioncomprising the polymers of the present invention, along with a carrieror diluent. The pharmaceutical composition can be used for therapy, suchas in the treatment of a disorder described herein. Similarly, theinvention provides for the use of a polymer disclosed herein as amedicament and for the use of a polymer disclosed herein in themanufacture of a medicament for the treatment of a disorder describedherein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the mucin complexing assay.

FIG. 2 illustrates the results of an oral glucose tolerance test onvehicle control (0.9% Saline) and treatment groups treated with 40 mg/mLof Example-3 and 80 mg/mL of Example-3 respectively. FIG. 2 depicts theblood glucose levels over a course of 120 minutes from each groupfollowing glucose administration.

FIG. 3 compares the reduction in the incremental area under the bloodglucose curve (iAUC) for the vehicle control (0.9% Saline) and treatmentgroups treated with 40 mg/mL of Example-3 and 80 mg/mL of Example-3respectively.

DETAILED DESCRIPTION OF THE INVENTION

This disclosure relates to cationic polymers that contain pendantsubstituted phenyl boronic acid groups, which are bonded directly orindirectly to the polymer backbone through an amine or amide bond. Thecationic polymers are preferably polycations that include amine- orammonium containing repeat units, and if desired may contain othercationic groups such as imidazolyl, pyridinyl, and guanidino. Thesubstituted phenyl boronic acid polymers preferably contain bothcationic repeat units and substituted phenyl boronic acid repeat units.The pendant phenyl boronic acid moiety is substituted with one or moresuitable substituents, which include electron withdrawing groups,electron donating groups, as described herein. In some embodiments thepolymers may contain a repeat unit that contains a cationic group and asubstituted phenyl boronic acid group. The cationic polymers can beco-polymers that also contain any desired neutral or anionic repeatunits, as further described herein, provided that the polymer retains anet cationic charge.

Pharmaceutical compositions comprising these polymers and methods oftreatment using these polymers to treat metabolic disorders, such asType-2 diabetes mellitus (T2DM), Type-1 diabetes mellitus (T1DM),prediabetes, hyperlipidemia, obesity, overweight, metabolic syndrome,non-alcoholic steatohepatitis, non-alcoholic fatty liver, and polycysticovary syndrome (PCOS) are also disclosed.

Exemplary polymers comprise a substituted phenyl boronic acidmoiety-containing repeat unit of Formula (I)-(III).

In Formulas (I) through (III):

R¹, R², R³ and R⁴ are independently hydrogen or substituted orunsubstituted alkyl;

Y¹ in each occurrence is independently a direct bond or -L¹-A¹-L²-A²-;

L¹ in each occurrence is —NR⁹—, —NC(O)— or —C(O)N—;

L² in each occurrence is absent, —NR⁹—, —O— or —S—;

Y² in each occurrence is independently a direct bond or -L³-A¹-L²-A²-;

L³ in each occurrence is —C(O)— or absent;

L² in each occurrence is absent, —NR⁹—, —O— or —S—;

A¹ and A² in each occurrence are independently absent or optionallysubstituted C₁-C₅ alkylene;

R⁹, R¹⁰ and R¹¹ in each occurrence are independently hydrogen orsubstituted or unsubstituted alkyl (preferably a substituted orunsubstituted C₁-C₆ alkyl);

Z in each occurrence is

preferably the —B(OH)₂ is at the 3- or 4-position of the phenyl ring;

-   -   X¹ and X² are independently selected from a group consisting of        hydrogen, halo, —CN, —NO₂, —N⁺(R⁹)(R¹⁰)(R¹¹), —CF₃, —SO₃(R⁹),        —SO₂(R⁹), —CON(R⁹)(R¹⁰), —(CH₂)_(m)—N(R⁹)(R¹⁰), and —OR⁹;    -   n is an integer from 1 to 100,000 and    -   m is an integer from 0 to 4;    -   with the proviso that no more than one of X¹ and X² is hydrogen;    -   when either X¹ or X² is —(CH₂)_(m)—N(R⁹)(R¹⁰), it is bonded to        the carbon atom in the phenyl ring that is adjacent to the        carbon atom in the phenyl ring to which —B(OH)₂ is bonded, and        the other of X¹ and X² is hydrogen.

In some embodiments of Formulas (I)-(III), X¹ is halo, —CN, —NO₂,—N⁺(R⁹)(R¹⁰)(R¹¹), —CF₃, —SO₃(R⁹), —SO₂(R⁹), or —CON(R⁹)(R¹⁰), and X² isnot —(CH₂)_(m)—N(R⁹)(R¹⁰) or —OR⁹; or X¹ is —(CH₂)_(m)—N(R⁹)(R¹⁰) or—OR⁹, and X² is not halo, —CN, —NO₂, —N⁺(R⁹)(R¹⁰)(R¹¹), —CF₃, —SO₃H, or—CON(R⁹)(R¹⁰).

In some preferred aspects, the polymer contains a repeat unit of Formula(I).

Preferred repeat units of Formula (I) include repeat units of Formulas(Ia)-(If):

In Formulas (Ia) through (If), R¹, R², R³, A¹, A², X¹, X², m and n areas described in Formula (I). In some embodiments, the —B(OH)₂ is at the3-position of the phenyl ring. In other embodiments, the —B(OH)₂ is atthe 4-position of the phenyl ring.

In some particular examples, the polymer includes a repeat unit ofFormula (Ia), (Ib), (Ic), (Id), (Ie) or (If), wherein R¹, R² and R³ areeach hydrogen.

In other particular examples, the polymer includes a repeat unit ofFormula (Ia), (Ib), (Ic), (Id), (Ie) or (If) wherein R¹ and R² are eachhydrogen, and R³ is alkyl, and preferably R³ is methyl.

It is advantageous to have substituents such as halo, preferably fluoro,—CN, —NO₂, —N⁺(R⁹)(R¹⁰)(R¹¹), —CF₃, —SO₃(R⁹), —SO₂(R⁹), and—CON(R⁹)(R¹⁰) on the phenyl boronic acid ring as these groups areelectron withdrawing by nature and can lower the pKa of the boronicacid.

It is also advantageous to have substituents such as—(CH₂)_(m)—N(R⁹)(R¹⁰) preferably —(CH₂)—N(Me)₂, on the phenyl boronicacid ring adjacent to the boronic acid group, as this group is electrondonating by nature and lowers the pKa value due to the interactionbetween B and N atoms.

Without wishing to be bound by any particular theory, it is believedthat certain substituted phenyl boronic acid polymers have a lower pKathat unsubstituted phenyl boronic polymers which lead to greatersensitivity to the pH increase between the stomach and duodenum,enhancing the mucin-complexing activity of the polymers at the desiredsite of barrier formation.

In Formulas (I) through (III), R¹, R², R³, R⁴, Y¹, Y², m, n and Z are asdescribed above, preferably the —B(OH)₂ is at the 3- or 4-position ofthe ring and X¹ and X² are at other positions of the ring.

In Formulas (I) through (III), R¹, R², R³, R⁴, Y¹, Y², m, n and Z are asdescribed above, preferably the —B(OH)₂ is at the 3-position of the ringand X¹ and X² are at other positions of the ring.

In Formulas (I) through (III), R¹, R², R³, R⁴, Y¹, Y², m, n and Z are asdescribed above, preferably the —B(OH)₂ is at the 4-position of the ringand X¹ and X² are at other positions of the ring.

In Formulas (I) through (III), R¹, R², R³, R⁴, Y¹, Y², m, n and Z are asdescribed above, preferably the —B(OH)₂ is at the 3- or 4-position ofthe ring and either X¹ or X² is —(CH₂)_(m)—N(R⁹)(R¹⁰), and X² is bondedto the carbon atom in the phenyl ring that is adjacent to the carbonatom in the phenyl ring to which —B(OH)₂ is bonded, and the other of X¹and X² is hydrogen.

In Formulas (I) through (III), R¹, R², R³, R⁴, Y¹, Y², m, n and Z are asdescribed above, preferably the —B(OH)₂ is at the 3-position of the ringand either X¹ or X² is —(CH₂)_(m)—N(R⁹)(R¹⁰), and X² is bonded to thecarbon atom in the phenyl ring that is adjacent to the carbon atom inthe phenyl ring to which —B(OH)₂ is bonded, and the other of X¹ and X²is hydrogen.

In Formulas (I) through (III), R¹, R², R³, R⁴, Y¹, Y², m, n and Z are asdescribed above, preferably the —B(OH)₂ is at the 4-position of the ringand either X¹ or X² is —(CH₂)_(m)—N(R⁹)(R¹⁰) that is bonded to thecarbon atom in the phenyl ring that is adjacent to the carbon atom inthe phenyl ring to which —B(OH)₂ is bonded, and the other of X¹ and X²is hydrogen.

In Formulas (I) through (III), R¹, R², R³, R⁴, Y¹, Y², m, n and Z are asdescribed above, preferably the —B(OH)₂ is at the 3-position of thering, X¹ is at the 5-position of the ring and is halo, preferablyfluoro, and X² is hydrogen, halo, —CN, —NO₂, —N⁺(R⁹)(R¹⁰)(R¹¹), —CF₃,—SO₃(R⁹), —SO₂(R⁹), or —CON(R⁹)(R¹⁰) at the 2-, 5- or 6-position of thering.

In Formulas (I) through (III), R¹, R², R³, R⁴, Y¹, Y², m, n and Z are asdescribed above, preferably the —B(OH)₂ is at the 4-position of thering, X¹ is at the 2-position of the ring and is halo, preferablyfluoro, and X² is hydrogen, halo, —CN, —NO₂, —N⁺(R⁹)(R¹⁰)(R¹¹), —CF₃,—SO₃(R⁹), —SO₂(R⁹), or —CON(R⁹)(R¹⁰) at the 3-, 5- or 6-position of thering.

In Formulas (I) through (III), R¹, R², R³, R⁴, Y¹, Y², m, n and Z are asdescribed above, preferably the —B(OH)₂ is at the 3-position of thering, X¹ is at the 5-position of the ring and is halo, preferablyfluoro, and X² is hydrogen.

In Formulas (I) through (III), R¹, R², R³, R⁴, Y¹, Y², m, n and Z are asdescribed above, preferably the —B(OH)₂ is at the 4-position of thering, X¹ is at the 2-position of the ring and is halo, preferablyfluoro, and X² is hydrogen.

In Formulas (I) through (III), R¹, R², R³, R⁴, Y¹, Y², m, n and Z are asdescribed above, preferably the —B(OH)₂ is at the 4-position of thering, X¹ is at the 2-position of the ring and is halo, preferablyfluoro, and X² is halo, preferably fluoro at 3-, 5- or 6-position of thering.

In Formulas (I) through (III), R¹, R², R³, R⁴, Y¹, Y², m, n and Z are asdescribed above, preferably the —B(OH)₂ is at the 4-position of thering, X¹ is at the 2-position of the ring and is halo, preferablyfluoro, and X² is halo, preferably fluoro at 3-position of the ring.

In Formulas (I) through (III), R¹, R², R³, R⁴, Y¹, Y², m, n and Z are asdescribed above, preferably the —B(OH)₂ is at the 4-position of thering, X¹ is at the 2-position of the ring and is —CF₃ and X² ishydrogen.

In Formulas (I) through (III), R¹, R², R³, R⁴, Y¹, Y², m, n and Z are asdescribed above, preferably the —B(OH)₂ is at the 3-position of thering, either X¹ or X² is —(CH₂)_(m)—N(R⁹)(R¹⁰) that is bonded to thecarbon atom in the phenyl ring that is adjacent to the carbon atom inthe phenyl ring to which —B(OH)₂ is bonded, and the other of X¹ and X²is hydrogen.

In Formulas (I) through (III), R¹, R², R³, R⁴, Y¹, Y², m, n and Z are asdescribed above, preferably the —B(OH)₂ is at the 4-position of thering, either X¹ or X² is —(CH₂)_(m)—N(R⁹)(R¹⁰) that is bonded to thecarbon atom in the phenyl ring that is adjacent to the carbon atom inthe phenyl ring to which —B(OH)₂ is bonded, and the other of X¹ and X²is hydrogen.

In Formulas (Ia) through (If), R¹, R², R³, A¹, A², m and n are asdescribed in Formula (I), preferably —B(OH)₂ is at 4-position of thering, X¹ is at the 2-position of the ring and is halo, preferablyfluoro, and X² is hydrogen.

In Formulas (Ia) through (If), R¹, R², R³, A¹, A², m and n are asdescribed in Formula (I), preferably —B(OH)₂ is at 4-position of thering, X¹ is a halo, preferably fluoro at 2-position of the ring and X²is halo, preferably fluoro at 3-position of the ring.

In Formulas (Ia) through (If), R¹, R², R³, A¹, A², m and n are asdescribed in Formula (I), preferably —B(OH)₂ is at 4-position of thering, X¹ is —CF₃ at 2-position of the ring and X² is hydrogen.

In Formulas (Ia) through (If), R¹, R², R³, A¹, A², m and n are asdescribed in Formula (I), preferably —B(OH)₂ is at 3-position of thering, X¹ is —(CH₂)_(m)—N(R⁹)(R¹⁰), preferably —CH₂—N(Me)₂ at 4-positionof the ring and X² is hydrogen.

In Formulas (Ia) through (If), R¹, R², R³, A¹, A², m and n are asdescribed in Formula (I), preferably —B(OH)₂ is at 4-position of thering, X¹ is —(CH₂)_(m)—N(R⁹)(R¹⁰), preferably —CH₂—N(Me)₂ at 3-positionof the ring and X² is hydrogen.

In other preferred aspects, the polymer contains a repeat unit ofFormula (II). In some examples of polymers that contain a repeat unit ofFormula (II), Y² is a direct bond and Z is

preferably the —B(OH)₂ is at the 3- or 4-position of the ring and X¹ andX² are as described above.

In other preferred aspects, the polymer contains a repeat unit ofFormula (II). In some examples of polymers that contain a repeat unit ofFormula (II), Y² is direct bond and Z is

preferably the —B(OH)₂ is at the 3- or 4-position of the ring and X¹ andX² are as described above.

In other preferred aspects, the polymer contains a repeat unit ofFormula (III). In some examples of polymers that contain a repeat unitof Formula (III), Y² is direct bond and Z is

preferably the —B(OH)₂ is at the 3- or 4-position of the ring and X¹ andX² are as described above.

The polymers can be homopolymers. When homopolymers, the polymerscontain a nitrogen-containing repeat unit (e.g. is a polyamine orpolyamide) with pendant boronic acid moieties bonded to the polymerbackbone directly or indirectly through the nitrogen atom of the repeatunit. Accordingly, the polymers typically contain secondary or tertiaryamines, or quaternary ammonium if desired, to which the boronic acidmoiety is bonded. The secondary or tertiary amines will be protonated atabout pH 5-7, providing cationic polymers.

Preferably, the polymers are copolymers that contain a repeat unit ofany one for Formulas (I)-(III) one or more other repeat units. The otherrepeat units are preferably cationic (e.g., a nitrogen-containing repeatunit), but can be neutral or anionic, provided that the polymer retainsan overall cationic charge.

Preferred nitrogen-containing repeat units that can be modified toinclude pendant boronic acid moieties include poly(allylamine) (PAAn),poly(diallylamine) (PDAAn), poly(ethyleneimine) (PEI) andpoly(methacrylamidopropylamine) (PMAPAn).

In the polymers disclosed herein, at least about 5% of the repeatingchemical units contain a pendant boronic acid group, e.g., a repeat unitof any one of Formulas (I)-(III). In some instances, substantially allof the chemical repeat units in the polymer contain a pendant boronicacid group. Preferably, about 5% to about 50%, about 5% to about 40%,about 5% to about 30%, about 5% to about 20%, or about 5% to about 15%of the repeating chemical units contain a pendant boronic acid group.

Suitable nitrogen-containing repeat units for inclusion in the polymersare well-known in the art, and include for example, polyvinylamine,poly-N-alkylvinylamine, polyacrylamide, polyalkylacrylamides (e.g.polymethacrylamides), poly-N-alkylacrylamides,polyalkyl-N-alkylacrylamides, polyallylamine, poly-N-alkylallylamine,polydiallylamine, poly-N-alkyldiallylamine, polyethylenimine,polyaminostyrene, polyvinylimidazole, polyvinylpyridine, and the like.

Nitrogen-containing repeat units are cationic when the amino nitrogen isprotonated. If desired the cationic character can be altered using knownmethods, such as, by converting amines into guanidino, biguanide,aromatics such as imidazolyl and pyridinyl, quaternary ammoniums, or byintroducing additional amino groups e.g., by alkylating an amine with analkylamino or alkylammonium group.

Polyamines typically are highly charged at duodenal pH (about pH 5-6),however, due to a high density of protonated amine sites in closeproximity, they deprotonate to a small extent as they pass from thestomach (pH ˜2) to the duodenum (pH ˜5) following ingestion. Even asmall amount of neutralization effectively lowers the polymer chargedensity and causes these polymer chains become more coiled, compact, andless well hydrated as pH is increased. Without wishing to be bound bytheory, this pH responsiveness is believed to contribute to preferentialcomplexing of the mucus in the duodenum over the stomach. Other polymersof this invention which are capable of responding to the pH increase ofthe duodenum contain cationic repeat units (e.g., repeat units withprotonated amines) that have inductive or structural features resultingin a lower pKa value than that of a standard protonated aliphatic amine.The lower pKa of these protonated polymers results in a greatersensitivity to the pH increase coincident with transit from the stomachto the duodenum. These types of polymers can therefore be targeted tointeract with the loose mucus of the proximal duodenum, and includepolyamines substituted with polar groups, such as hydroxyl groups lessthan three carbon atoms away from the protonated amine. In someembodiments, the polymers include amines that are modified to have alower pKa than the unmodified amines. For example, the cationic polymerscan have pKa values less than 9.0, more preferably a pKa less than 8.0,and most preferably a pKa less than or equal to 7.0.

Suitable boronic acid containing repeat units for inclusion in thepolymers described herein include, but not limited to, for example,repeat units of Formulas (I), (Ia)-(If), (II) and (III), including thefollowing:

where “m” represents an integer from 1 to 100,000 and

-   -   X¹ and X² are described as above.

Exemplary polymers that contain substituted phenyl boronic acid repeatunits include the following:

Exemplary polymers that contain substituted phenyl boronic acid repeatunits include the following:

Suitable nitrogen-containing cationic monomeric repeat units include,but not limited to, for example, the following:

where “n” represents an integer from 1 to 100,000.

If desired, the cationic polymers that contain pendant boronic acidgroups can also include a hydrophobic group, e.g., a pendent hydrophobicgroup. As used herein hydrophobic groups are moieties that are moresoluble in octanol than water (as a separate chemical entity). Forexample, an octyl substituent is a hydrophobic group because octane ismore soluble in octanol than in water. Suitable hydrophobic groups areC6 or greater linear, branched or cyclic hydrocarbons that can besubstituted, for example with one or more hydroxy, halo, and/or aryl(e.g., benzyl) groups.

Additional repeat units that can be included in the polymers describedherein, when desired, include neutral and acid repeat units, such as,polyacrylates, polyalkylene glycols, polystyrene, polyvinyl alcohols,polyvinylphosphates, polyvinylsulfates, and the like.

Particular examples of polymers of this disclosure include

Additional, particular examples of polymers of this disclosure include

Additional particular examples of polymers of this disclosure include

Additional particular examples of polymers of this disclosure include

Additional particular examples of polymers of this disclosure include

Copolymers of the present invention can exist in a variety of forms.Suitable forms include block copolymers, graft copolymers, combcopolymers, star copolymers, dendrimers, hyperbranched copolymers,random copolymers, gradient block copolymers, and alternate copolymers.

This disclosure also relates to cationic polymers that contain pendanthydrophobic groups, and to the use of such polymers for treatingmetabolic disease as disclosed herein.

Preferably, the polymers disclosed herein are of sufficient size so thatthe polymers are substantially not absorbed when administered orally toa subject, such as a human. The threshold molecular weight above whichpolymers are not absorbed from the GI tract into the systemiccirculation is dependent on the specific polymer and conditions in theGI tract and other factors, but it is generally recognized that polymersof greater than 1,000 Da are not substantially absorbed from the GItract into the systemic circulation. Accordingly, the compositions ofthis invention that are substantially not absorbed from the GI tract aresubstantially free of polymer chains smaller than 1,000 Da, andpreferably 5,000 Da or more preferably 10,000 Da and have averagemolecular weights (M_(w)) of at least about 10,000 Da and preferably inthe range of 20,000 to 250,000 Da or greater. The polymers of theinvention can contain a distribution of polymer chain lengths, and mayhave a polydispersity index (PDI) in the 1.5-4.0 range, but they containsubstantially no material under 1,000 Da, preferably they contain nomaterial under 5,000 Da or more preferably under 10,000 Da.

The inventive polymers are soluble and are preferably not cross-linked.In some embodiments the polymers may be lightly cross-linked but remainsoluble and do not form an extended network or gel.

Also included in the present invention are pharmaceutically acceptablesalts of the disclosed polymers. For example, polymers which have acidfunctional groups can also be present in the anionic, or conjugate base,form, in combination with a cation. Suitable cations include alkalineearth metal ions, such as sodium and potassium ions, alkaline earthions, such as calcium and magnesium ions, and unsubstituted andsubstituted (primary, secondary, tertiary and quaternary) ammonium ions.Polymers which have basic groups such as amines can also be protonatedand have a pharmaceutically acceptable counter anion, such as halides(Cl⁻ and Br⁻), CH₃OSO₃ ⁻, HSO₄ ⁻, SO₄ ²⁻, HCO³⁻, CO₃ ²⁻, nitrate,hydroxide, persulfate, sulfite, acetate, formate, sulfate, phosphate,lactate, succinate, propionate, oxalate, butyrate, ascorbate, citrate,dihydrogen citrate, tartrate, taurocholate, glycocholate, cholate,hydrogen citrate, maleate, benzoate, folate, an amino acid derivative, anucleotide, a lipid, or a phospholipid. Similarly, ammonium groupscomprise a pharmaceutically acceptable counteranion. Boronic acid groupscan react with anions such as sodium or potassium hydroxide, alkoxide orcarboxylate to form a salt such as B—(OH)₃Na⁺, B—(OH)₃K⁺,B—(OH)₂(OCH₃)Na⁺, B—(OH)₂(OCH₃)K⁺, B—(OH)₂(OCOCH₃)Na⁺,B—(OH)₂(OCOCH₃)K⁺, and the like.

The polymers disclosed herein are typically provided as a mixture ofpolymer chains with some variability in chain length. This distributionof polymer chain lengths can be measured using size exclusionchromatography (SEC) and a detector capable of measuring polymer molarmass such a multi-angle laser light scattering (MALLS). This method canalso confirm the absence of short, low molecular weight polymer chains.It can also provide a polydispersity index (PDI), which is typicallyconsidered to be the ratio M_(w)/M_(n), where M_(w) is the weightfraction-average molecular weight and M_(n) is the number averagemolecular weight.

PDI=M _(w) /M _(n)

Values of M_(w), M_(n) and PDI can be obtained by SEC, preferable with aMALLS detector. For synthetic polymer materials made from standardfree-radical processes, it is common to find PDI values greater than 2,and even greater than 3. In contrast, living free radical polymerizationprocesses such as atom transfer radical polymerization (ATRP) orreversible addition fragmentation chain transfer (RAFT) are capable ofproducing materials with PDI less than 2, or even less than 1.5.

The polymers disclosed herein can be prepared using any suitablemethods, such as by direct polymerization of one, two or more monomersor by polymer modification.

Polymerization can be accomplished using techniques known in the art ofpolymer synthesis (See, for example, Shalaby et al, ed., Water-SolublePolymers, American Chemical Society, Washington, D.C. [1991]). Severalcationic monomers are available as hydrochloride salts and can bepolymerized by methods known in the art, for example, via a free radicaladdition process. In this case, the polymerization mixture includes afree-radical initiator. Suitable free-radical initiators includeazobis(isobutyronitrile), azobis(4-cyanovaleric acid),2,2′-azobis(2-amidinopropane)dihydrochloride, potassium persulfate,ammonium persulfate, and potassium hydrogen persulfate. Other suitableinitiators include ionizing radiation and ultraviolet light. The freeradical initiator is preferably present in the reaction mixture in anamount ranging from about 0.01 mole percent to about 5 mole percentrelative to the monomer.

Polymer modification approach employs polyamines and copolymer approachemploys acrylamide derivatives. “M” in the reaction schemes represents agroup that includes a substituted phenyl boronic acid moiety.

Polyamines can serve as mucus-interacting agents as well as startingmaterials for chemical modification with boronic acid groups. Exemplarypolyamines include polyethyleneimine, hydroxyethylatedpolyethyleneimine, polyamidoamine (PAMAM) dendrimers, poly(allylamine)(PAAn) and its copolymers, poly(diallylamine) (PDAAn) and itscopolymers, poly(vinylamine) and its copolymers, poly(vinylimidazole)and its copolymers, poly(vinylpyridine) and its copolymers,poly(vinylaniline) and its copolymers, amine containing acrylamide andmethacrylamide copolymers, and the like. Preferred polyamines includepoly(allylamine) (PAAn), poly(diallylamine) (PDAAn), poly(ethyleneimine)(PEI) and poly(methacrylamidopropylamine) (PMAPAn).

Polyamine derivatives can be obtained from the chemical modification ofpolyamines by amide-forming chemistry using EDC coupling (Scheme 1).

A 1% wt/vol of desired polyamine is prepared in deionized water the pHis adjusted to 5.0. Ethanol or other suitable organic is added to thepolymer solution at 50% of initial polymer solution volume. TheM-carboxylic acid to be coupled is placed into water at 25% of initialpolymer solution volume to form a solution or slurry. The EDC couplingagent is dissolved in ethanol or other suitable solvent at 25% ofinitial polymer solution volume. The EDC solution is then mixed with theM-carboxylic acid solution or slurry. The combined EDC/M-carboxylic acidsolution is added to the polymer solution dropwise by pipette orpressure equalizing addition funnel, over approximately 10 minutes. Thereaction solutions contains polymer at about 0.5% wt/vol with about 62%vol water and about 38% vol ethanol or other suitable organic solvent.The reaction is stirred and pH is maintained at 5.0. With pH stabilizedat 5.0, the reaction is allowed to stir at room temperature for about 18hours. The polymer is precipitated with excess (3× volume) acetone.

The polyamine derivatives can be obtained from the chemical modificationof polyamines by Michael addition reaction. The polyamine is thenucleophile and the acrylamides are the Michael acceptor (Scheme 2).

A 1% wt/vol of desired polyamine is prepared in deionized water and thepH was adjusted to 8.5. This pH can be increased or lowered depending onlevel of modification desired. The desired M-acrylamide is dissolved inethanol or other suitable solvent at 20% of initial polymer solutionvolume. The acrylamide solution is then added to the polymer solution toform a reaction mixture with the polymer at about 0.83% wt/vol withabout 83% vol water and about 17% vol ethanol or other suitable solvent.The reaction mixture is heated to 70° C. and stirred for 48 hours. Thepolymer is precipitated with excess (3× volume) acetone.

The polyamine derivatives can be obtained from the chemical modificationof polyamines by hydroxyalkylation using epoxide-opening chemistry(Scheme 3).

A 2% wt/vol of desired polyamine is prepared in deionized water and thepH is adjusted to 6.0. This pH can be increased or lowered depending onlevel of modification desired. The desired M-epoxide is dissolved inwater/ethanol (25%/75%) at 100% of initial polymer solution volume. Thissolution is then added to the polymer solution to prepare a reactionsolution in which the polymer was about 1% wt/vol with about 62% volwater and about 38% vol ethanol. The reaction mixture was heated at 60°C. for 48 hr. If the epoxide is not fully in solution at 60° C., a smallportion of additional ethanol may be added to aid in solubility. Thepolymer is precipitated with excess (3× volume) acetone.

Polyacrylamide derivatives can be obtained, for example, by thepolymerization of 3-acrylamidopropylamine with a desired M-acrylate orM-acrylamide (Scheme 4).

A desired amount of the desired M-acrylate or M-acrylamide monomer isplaced into a 30 ml glass vial equipped with magnetic stirring and an N₂(g) inlet. The desired M-acrylate or M-acrylamide monomer is dissolvedin dimethylformamide or other suitable water-miscible organic solvent.The desired amount of cationic, neutral or anionic co-monomer is thenadded. A small amount of water will likely be needed to fully dissolvethe charged co-monomer in a binary solvent system. An appropriate amountof AIBN initiator is added. The co-monomer solution is N₂ (g) purged forat least ˜15 minutes. The reaction is then heated at 65° C. while undera blanket of N₂ (g). After several hours of heating, the copolymersolution or suspension is isolated by precipitation from acetone. Theaddition of some water and adjustment to lower pH will be needed forsome of the polymers to facilitate precipitation in acetone. Finally,the product may be dissolved in deionized water, IPA/dry ice frozen andlyophilized.

Accordingly, in some aspects, the invention provides a method forapplying a physical barrier to the gastrointestinal (GI) tract of asubject between the intestinal lining and the luminal contents. Themethod includes administering to the GI tract of the subject atherapeutic effective amount of a polymer described herein.

As used herein, the term “physical barrier” or “luminal barrier” refersto a complex of polymer and mucus that prevents or reduces chyme fromcontacting the mucosal epithelium located under the polymer-mucuscomplex in the intestinal tract. The physical barrier is created whenthe polymer combines in-situ with the anionic mucins contained withinthe mucus lining the wall of the intestines. The physical barrier can besubstantially complete or partial. A substantially complete physicalbarrier extends to substantially cover an entire target area, such asthe epithelial lining of the proximal duodenum. A partial physicalbarrier extends to cover a portion of a target area, such as a portionof the epithelial lining of the duodenum. For example, a partial barriercan cover at least about 1%, 5%, 10%, 20%, 25%, 30%, 40%, 50% or more ofthe epithelium at the target site.

The partial physical barrier can be discontinuous and spatiallydistributed, and may have varying degrees of permeability. For instance,physical barrier can be a semi-permeable complex of polymer and mucus ormucins on the luminal surface of the intestines, preferably in theduodenum.

In embodiments, the physical barrier or a formulation thereof is passedby natural digestive processes of the subject. In yet other embodiments,the physical barrier is removable or reversible by the ingestion of aliquid or solvent.

The polymers described herein bind tightly to the mucus and mucins toform a polymer-mucus complex that is resistant to disassociation (e.g.,by high salt and low pH). Accordingly, once formed, the physical barrierwill typically be present for a “retention period” or “residence time,”and is removed by the natural actions of the digestive system. Typicalretention periods can range from about half an hour to about 7 days,including time period ranging from about 1 hour to about 3 hours, about1 hour to about 5 hours, about 1 hour to about 24 hours, about 1 toabout 3 days, and others.

The desired residence time can vary depending on the clinicalapplication and can be adjusted based on the amount of polymer that isadministered, and the frequency and interval between administrations.For instance, up to 50% of subjects with T2DM have gastroparesis, ordelayed gastric emptying, that may require the mucoadhesive lining toremain in place for a longer time than a pre-diabetic or non-diabeticobese subject. Blood glucose levels spike often within the first twohours of eating a meal, most often within the first 60 minutes; thus,the lining should adhere for a minimum of 60 minutes in one embodiment.In another embodiment, in the case of pre-diabetic subjects who may nottake medication prior to every meal, and thus may not comply with atreatment that would require to change their behavior, a longer lastingmucoadhesive lining may be required. In this application, the lining mayadhere for a minimum of 6-8 hours with a maximum of 24 hours could berequired. Residence time will also be influenced by the mucus layer atwhich the polymer develops the most affinity to. For instance, thesuperficial, loosely adherent layer sloughs off on the order of minutesto hours, whereas affinity to the deeper firmly adherent layer wouldlead to a longer lasting mucoadhesive coating. Overall, residence timecan be tuned to various clinical and technical considerations in theembodiments outlined in this disclosure.

Polymers of the invention can form an occlusive barrier layer in theproximal intestine, specifically the duodenum. Preferably, the occlusivebarrier is formed in the proximal duodenum or duodenal bulb. Thepolymers are therefore fully capable of forming a barrier layerimmediately upon release from the stomach and entry into the proximalduodenum.

The polymers are administered orally in any suitable dosage form. Avariety of dosage forms that are suitable for oral administration arewell-known in the art and include, liquid formulations (e.g., solutions,suspensions, slurrys, syrups), gels, ointments, powder, tablets,caplets, capsules and the like.

In one example, the polymers can be administered in a liquid form andare typically sufficiently stable and soluble in the stomach allowingimmediate delivery to the duodenum in an active state without requiringfurther swelling, solubilization, or equilibration with the surroundingmilieu. The polymers described herein are typically polyamines, whichundergo some degree of deprotonation as they transition from the highlyacidic stomach (pH˜2) to the duodenum (pH ˜5) which targets thecomplexing activity of the polymers to the duodenum. However, thepolymers may also form a barrier layer in the stomach.

In other examples, the polymer is administered in a solid form capableof being hydrated in the stomach. The solid form can be formulated toprovide slow dissolution, which can protect the polymers from gastricacidity, but resulting in the polymers entering the proximal duodenum ina fully active state. In other examples, the polymers are administeredin the form of an enteric-coated tablet, caplet, capsule or otherenteric-coated dosage form to protect the polymers from gastric acidity.In such examples, the enteric coating is formulated to dissolve ordegrade as soon as possible after or during passage through the pyloricvalve (when the pH increases from pH-2) permitting the immediate releaseof the polymers. Such dosage forms can include a superdisintegrant tofacilitate immediate release of the polymers at the desired site in theintestinal tract, such as the proximal duodenum. Suitablesuperdisintegrant excipients are well-known in the art. (See, e.g.,Mohanachandran, P. S. et al, Superdisintegrants: An Overview, Int. J.Pharma. Sci. Review and Research, (2011) 6:1 pp 105-109.) For example,enteric capsules have been described in the literature that are capableof targeting delivery to the duodenum. (See, e.g., Reix N. et al. Intl JPharm (2012) 422:1-2 pp. 338-340.)

The polymers of the invention can quickly dissolve after oraladministration in the stomach, in the duodenum or on other mucosalsurfaces.

In some aspects, the pharmaceutical formulations of the polymers of thisinvention may optionally include a calcium salt, such as calciumchloride or calcium citrate. It is believed that the physical barrierformation can be accelerated in the presence of a calcium salt.

If desired, the polymers can be administered to the gastrointestinaltract of the subject via an endoscope, a nasal feeding tube, an oralfeeding tube, or similar device. The polymer can also be sprayed ontothe mucosa at the desired site of action, for example, the spraying canbe done endoscopically.

For therapeutic purposes, a “therapeutically effective amount” of thepolymer is administered. A therapeutically effective amount, as usedherein is an amount sufficient to affect the desired response under theconditions of administration, including clinical response. Thetherapeutically effective amount may be sufficient, for example, toimprove glucose homeostasis, to reduce insulin resistance, to causeweight loss, and/or to improve other signs and/or symptoms of T1DM, T2DMor other metabolic disorders such as hyperlipidemia, non-alcoholicsteatohepatitis, non-alcoholic fatty liver and other conditions such asobesity and overweight. For example, a therapeutically effective amountcan be an amount sufficient to lower blood glucose levels and/or reduceHbA1C.

The precise amount that is administered will depend on a number ofwell-known considerations, including the age, weight, gender, particularcondition to be treated and its severity, sensitivity to drugs, andoverall health of the subject. A skilled clinician can determineappropriate amounts to administer based on these and otherconsiderations. Typically, 1 to 5 tablets/capsules are administered perdose, each of size 0 or 0E or 00 or 00E or 000. A dose can beadministered one, two, three or four times per day. The dose timing willbe based on the underlying indication. Preferably, a dose isadministered at least 5, 10, 15, 30 or 60 min and not more than 12 hrsbefore a meal for the treatment of metabolic conditions. For otherindications, dosing right before or along with food may be preferred.

As used herein and as well understood in the art, “treatment” is anapproach for obtaining beneficial or desired results, including clinicalresults. Beneficial or desired clinical results may include, but are notlimited to, alleviation or amelioration of one or more symptoms orconditions, diminution of extent of disease or affliction, a stabilized(i.e., not worsening) state of disease or affliction, preventing spreadof disease or affliction, delay or slowing of disease or afflictionprogression, amelioration or palliation of the disease or afflictionstate and remission (whether partial or total), whether detectable orundetectable. “Treatment” can also mean prolonging survival as comparedto expected survival if not receiving treatment.

This disclosure relates to methods of treating metabolic disease byadministering a therapeutically effective amount of a polymer disclosedherein to a subject in need thereof. Metabolic diseases that can betreated using the method include, for example, glucose intolerance,T1DM, T2DM, prediabetes, hyperlipidemia, obesity, overweight, obesity,dyslipidemia, hypertension, hyperglycemia, impaired glucose tolerance,insulin resistance, metabolic syndrome, non-alcoholic steatohepatitis(NASH), non-alcoholic fatty liver disease (NAFLD) and polycystic ovarysyndrome (PCOS).

This disclosure also relates to methods of treating gastrointestinaldisorders by administering a therapeutically effective amount of apolymer disclosed herein to a subject in need thereof. Gastrointestinaldisorders that can be treated using the method include, for example,celiac disease, irritable bowel syndrome, inflammatory bowel disease,colitis, Clostridium difficile, endotoxemia, diarrhea and constipation.

The methods of the invention are also useful in addressing leaky gutsyndrome and associated conditions. Leaky gut syndrome is a term of artthat describes a condition in which there is increased intestinalpermeability due to alteration/damage to the tight epithelial junctionswhich results in a compromised epithelial barrier function. Thisimpaired barrier acts as a conduit for intraluminal macromolecules andantigens to permeate through the gut wall triggering inflammatory,immunological reactions that result in various health conditions. Forexample, leaky gut syndrome has been implicated in IBS (irritable bowelsyndrome). Certain proteins in foods can behave as antigens eliciting animmune response. For example, in Celiac disease, preventing gluten fromcoming into contact with the epithelium can reduce the immunologicalresponse. Leaky gut has also been implicated in other immunologicalconditions like Inflammatory Bowel Disease (Crohn's disease, UlcerativeColitis). An enhanced intestinal barrier can reduce the absorption ofendotoxins in the gastrointestinal tract. Some of these endotoxins are aresult of normal bacterial metabolism/breakdown or due to bacterialovergrowth. This is particularly relevant in patients with impairedliver function, e.g., in liver cirrhosis, in whom the endotoxins are notmetabolized (detoxified) by the liver resulting in impaired brainfunction (called hepatic encephalopathy). Thus the methods describedherein can be used to enhance the barrier properties of the intestineand can treat or reduce the incidence of hepatic encephalopathy. Uremiais another condition associated with an impairment of intestinal barrierfunction that can be treated or reduced using the methods describedherein. Similarly, the methods described herein can be used to treat orreduce the incidence of Chronic Kidney Disease (CKD), as clinicalevidence has documented greater intestinal permeability in patients withadvanced CKD.

The therapeutic methods can also provide benefit by reducing theclinical biomarkers associated with a variety of disorders, such asreducing systemic inflammation, oxidative stress and hyperuricaemia.

The disclosed polymers can be administered to the subjects in the formof a pharmaceutical composition that includes a pharmaceuticallyacceptable carrier, excipient, buffer or diluent.

For oral administration, the pharmaceutical compositions of theinvention may be presented in dosage forms such as capsules, tablets,caplets, powders, granules, gels, suspensions, solutions or othersuitable dosage form. Capsule may be gelatin, soft-gel or solid. Tablet,caplet and capsule formulations may further contain one or moreadjuvants, binders, diluents, disintegrants, excipients, fillers, orlubricants, each of which are known in the art. Examples of such includecarbohydrates such as lactose or sucrose, dibasic calcium phosphateanhydrous, corn starch, mannitol, xylitol, cellulose or derivativesthereof, microcrystalline cellulose, gelatin, stearates, silicondioxide, talc, sodium starch glycolate, acacia, flavoring agents,preservatives, buffering agents, disintegrants, and colorants.

Orally administered compositions may contain one or more optional agentssuch as, for example, sweetening agents such as fructose, aspartame orsaccharin; flavoring agents such as peppermint, oil of wintergreen, orcherry; coloring agents; and preservative agents, to provide apharmaceutically palatable preparation. Suitable pharmaceuticalformulations for oral administration and methods for preparing them arewell-known in the art. See, e.g., Remington: The Science and Practice ofPharmacy, twentieth edition, 2000.

Pharmaceutical preparations that can be used orally include push-fitcapsules made of a suitable material, such as gelatin, as well as soft,sealed capsules made of a suitable material, for example, gelatin, and aplasticizer, such as glycerol or sorbitol. The push-fit capsules cancontain the active ingredients in admixture with filler such as lactose,binders such as starches, and/or lubricants such as talc or magnesiumstearate and, optionally, stabilizers. In soft capsules, the activecompounds can be dissolved or suspended in suitable liquids, such asfatty oils, liquid paraffin, or liquid polyethylene glycols. Inaddition, stabilizers can be added. All formulations for oraladministration should be in dosages suitable for such administration.Methods for encapsulating compositions (such as in a coating of hardgelatin or cyclodextran) are known in the art (Baker, et al.,“Controlled Release of Biological Active Agents”, John Wiley and Sons,1986).

The methods of the invention include a co-formulation of the polymericcomposition comprising with probiotics. Probiotic formulations assist inbuilding the beneficial probiotic bacteria in the intestinal tract. Itis known in the art that probiotics have significant effects on thereduction of blood sugar, HbAlc, insulin levels and insulin resistancein subjects with diabetes. Suitable probiotics include, but not limitedto Lactobacillus Bifidobacteria, Saccharomyces boulardii, and Bacilluscoagulans, Akkermansia muciniphila, Bifidobacterium spp, Escherichiaspp. Methods to prepare formulations containing probiotics are wellknown in the art.

If desired, the therapeutic methods described herein can includeco-administration of the polymeric compositions with one or moreadditional therapeutic agents. Therapeutic agents for co-administrationin subjects with diabetes may include classes of drugs that are GLP-1receptor agonists, DPP-4 inhibitors, SGLT-2 inhibitors, glucosidaseinhibitors, insulin, metformin, sulfonylureas and thiazolidenediones.

In particular examples, the additional therapeutic agent is one or moreagent indicated for the treatment of diabetes (type 1 and/or type 2),pre-diabetes, hyperglycemia, impaired glucose tolerance or insulinresistance. Such agents include biguanides (e.g., metformin),sulfonylureas (e.g., limepiride, gliclazide, gilpizide, glimepiride,tolbutamide, glibenclamide (glyburide), gliquidone, and glyclopyramide),meglinithinides (e.g., repaglinide and nateglinide); thiazolidindiones(e.g., pioglitazone and rosiglitazone), alpha-glucosidase inhibitors(e.g., acarbose and miglitol), dipeptidyl peptidase 4 (DPP4) inhibitors(e.g., vildagliptin, sitagliptin, saxagliptin, and linagliptin), GLP-1analogues (e.g., exenatide, lixisenatide, dulaglutide, and liraglutide),sodium-glucose co-transporter 2 (SGLT2) inhibitors (e.g., dapagliflozin,ganagliflozin, and empagliflozin), amylin mimetics (e.g., pramlinitide),D2-dopamine agonists (e.g., bromocriptine), bile acid sequestrants(e.g., cholestyramine, colesevelam, colestilan, and colestimide), andinsulin (e.g., human insulin, insulin glulisine, insulin lispro, insulinisophane human, insulin zinc suspension mixed bovine, insulin protaminezinc bovine, insulin isophane porcine, insulin isophane human, and thelike).

The co-therapeutic methods can provide several advantages overmonotherapy. For example, administering the polymer and an additionaltherapeutic agent can enhance the efficacy of and/or reduce the amountof additional therapeutic agent that is needed for the desired effect.Accordingly, undesired side effects of the additional therapeutic agentcan be reduced or eliminated. Additionally, the polymers of theinvention and the additional therapeutic can provide superior therapy incomparison to each agent as a monotherapy, and co-therapy can provideadditive or synergistic effects.

The subject to be treated by the presently disclosed methods istypically a mammal and preferably a human subject. Suitable subjectsinclude mammals including, but not limited to, primates, e.g., humans,monkeys, apes, and the like; bovines, e.g., cattle, oxen, and the like;ovines, e.g., sheep and the like; caprines, e.g., goats and the like;porcines, e.g., pigs, hogs, and the like; equines, e.g., horses,donkeys, zebras, and the like; felines, including wild and domesticcats; canines, including dogs; lagomorphs, including rabbits, hares, andthe like; and rodents, including mice, rats, and the like. Preferably,the subject is a human including, but not limited to, fetal, neonatal,infant, juvenile, and adult humans.

The terms “a,” “an,” and “the” refer to “one or more” when used in thisapplication, including the claims. Thus, for example, reference to “asubject” includes a plurality of subjects, unless the context clearly isto the contrary (e.g., a plurality of subjects), and so forth.

Throughout this specification and the claims, the terms “comprise,”“comprises,” and “comprising” are used in a non-exclusive sense, exceptwhere the context requires otherwise. Likewise, the term “include” andits grammatical variants are intended to be non-limiting, such thatrecitation of items in a list is not to the exclusion of other likeitems that can be substituted or added to the listed items.

Although specific terms are employed herein, they are used in a genericand descriptive sense only and not for purposes of limitation. Unlessotherwise defined, all technical and scientific terms used herein havethe same meaning as commonly understood by one of ordinary skill in theart to which this presently described subject matter belongs.

The term “about,” when referring to a value means±20%, or +10. Further,the term “about” when used in connection with one or more numbers ornumerical ranges, should be understood to refer to all such numbers,including all numbers in a range and modifies that range by extendingthe boundaries above and below the numerical values set forth. Therecitation of numerical ranges by endpoints includes all numbers, e.g.,whole integers, including fractions thereof, subsumed within that range(for example, the recitation of 1 to 5 includes 1, 2, 3, 4, and 5, aswell as fractions thereof, e.g., 1.5, 2.25, 3.75, 4.1, and the like) andany range within that range.

As used herein, the term ‘alkyl” refers to monovalent aliphatichydrocarbon typically containing 1 to about 6 carbon atoms. An alkylgroup can be straight chain, branched chain, monocyclic moiety orpolycyclic moiety or combinations thereof. Suitable substituents for analkyl group include aryl, OH, halogen (—Br, Cl, I and F), O(R′),O—CO—(R′), CN, NO₂, COOH, NH₂, —NH(R′), N(R′)₂, COO(R′), CONH₂,CONH(R′), CON(R′)₂, S(O)R′, S(O)₂R′, SH and S(R′). Each R′ isindependently an alkyl group or an aryl group. A substituted alkyl groupcan have more than one substituent. Examples of alkyl groups includemethyl, ethyl, propyl, isopropyl, butyl, iso-butyl, sec-butyl,tert-butyl, pentyl, hexyl, cyclopropyl, cyclobutyl, cyclopentyl,cyclohexyl, norbornl, and the like.

As used herein, the term ‘alkylene” refers to —(CH₂)_(x)—, that may beoptionally substituted, where x is an integer between 1 to 5.Preferably, x is between 1 to 3, more preferably x is 1 or 2. Suitablesubstituents for an alkylene group are identical to those for alkylgroups.

As used herein, the term “alkoxy” refers to a group of formula —O—alkyl. Example of alkoxy groups include methoxy, ethoxy, propoxy (e.g.,n-propoxy and isopropoxy), tert-butoxy, and the like.

As used herein, the term “aryl,” refer to stable aromatic monocyclicring system having 3-7 ring atoms, of which all the ring atoms arecarbon, and which may be substituted or unsubstituted. Aryl substituentsinclude, OH, halogen (—Br, Cl, —I and F), O(R′), —O—CO—(R′), CN, NO₂,COOH, NH₂, —NH(R′), N(R′)₂, COO(R′), CONH₂, CONH(R′), CON(R′)₂, S(O)R′,S(O)₂R′, —SH and S(R′). Each R′ is independently an alkyl group.

As used herein, the term “aryloxy” refers to a group of formula —O—aryl. The aryl group may optionally substituted.

As used herein, the term “electron withdrawing” refers to an atom or agroup that draws electron density from neighboring atoms towards itself,usually by resonance or inductive effects. Suitable electron withdrawinggroups include, but not limited to halo, —CN, —NO₂, —N⁺(R⁹)(R¹⁰)(R¹¹),—CF₃, —SO₃(R⁹), —SO₂(R⁹), and —CON(R⁹)(R¹⁰).

As used herein, the term “electron donating” refers to an atom or agroup that donates electron density to neighboring atoms, usually byresonance or inductive effects. Suitable electron donating groupsinclude, but not limited to alkyl, amino, —(CH₂)_(m)—N(R⁹)(R¹⁰), and—OR⁹.

As used herein, the term “hydroxy” refers to a group of formula —OH.

As used herein, “halo” or “halogen” refers to F, Cl, Br, or I.

As used herein, the phrase “optionally substituted” means unsubstitutedor substituted. As used herein, the term “substituted” means that ahydrogen atom is removed and replaced by a substituent. It is to beunderstood that substitution at a given atom is limited by valency.

The following examples are provided to further illustrate theembodiments of the present invention, but are not intended to limit thescope of the invention. While they are typical of those that might beused, other procedures, methodologies, or techniques known to thoseskilled in the art may alternatively be used.

EXAMPLES I. Synthesis:

Reagents: Poly(allylamine hydrochloride) was obtained from NittoboMedical, Japan (PAAn-HCl, Cat #PAA-HCl-3L, 50.3% solution in water) andused as received. The material was qualified by 1H-NMR, TGA, and sizeexclusion chromatography (SEC-MALLS).1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride was obtainedfrom Chem-Impex International, Wood Dale, Ill. (EDC-HCl, Cat#00050,99.8%) and used as received. The material was qualified by 1H-NMR,FT-IR, and melting point. 1-Hydroxybenzotriazole hydrate was obtainedfrom Chem-Impex International, Wood Dale, Ill. (HOBt, Cat#24755, 99.8%(odb), 21.3% water) and used as received. The material was qualified by1H-NMR, and FT-IR. 4-Carboxyphenylboronic acid was obtained fromChem-Impex International, Wood Dale, Ill. (CPBA, Cat#28086, 99.7%) andused as received. The material was qualified by 1H-NMR, FT-IR, andmelting point. 3-Trifluoromethyl-4-Carboxyphenylboronic acid wasobtained from Combi-Blocks Inc., San Diego, Calif. (CF3-CPBA, Cat#FA-2133, 98%) and used as received. The material was qualified by1H-NMR, and FT-IR. 3-Fluoro-4-Carboxyphenylboronic acid was obtainedfrom AOB Chem, Zhuhai, China (F-CPBA, Cat#10055, 97%) and used asreceived. The material was qualified by 1H-NMR, FT-IR, and meltingpoint. 2,3-Difluoro-4-Carboxyphenylboronic acid was obtained fromCombi-Blocks Inc., San Diego, Calif. (Di-F-CPBA, Cat #FA-3733, 96%) andused as received. The material was qualified by 1H-NMR, and FT-IR.3-Dimethylaminomethyl-4-Carboxyphenylboronic acid pinacol ester wasobtained from Combi-Blocks Inc., San Diego, Calif. (Me2N-CPBA, Cat#PN-4078, 96%) and used as received. The material was qualified by1H-NMR, and FT-IR

EXAMPLE 1. SYNTHESIS OF PAAn modified with 4-Carboxyphenylboronic acid:PAAn-HCl 50.3% solution (5.964 g, 32 mmol of amine equivalents) wasplaced in a 250 ml beaker with a magnetic stir bar and deionized water(90 ml). The resulting clear solution was magnetically stirred, and a pHelectrode introduced. The pH was adjusted to 8.0 by dropwise addition ofa 1N NaOH solution while stirring. 4-Carboxyphenylboronic acid, 99.7%(0.907 g, 5 mmol) was added to the reaction mixture and the resultingsuspension was stirred. After 20 minutes of stirring, the pH had droppedto 6.8 and some solid remained suspended in solution. Additional NaOHsolution was added in portions with stirring to cause the completedissolution of the suspension. The pH of the resulting clear solutionwas 7.5. Solid HOBt-hydrate powder (0.046 g, 0.25 mmol) was then addedto the clear reaction solution and dissolved after 20 minutes ofstirring. Hydrochloric acid (1N) was added to the reaction mixture tolower the pH to 5.4. The reaction solution remained clear. EDC-HCl(1.152 g, 6 mmol) dissolved in 10 ml of deionized water was then slowlypipetted into the reaction mixture. The clear reaction mixture had afinal volume of 120 ml and a pH of 5.6. This reaction mixture wasstirred for 18-hours. The reaction mixture was pH adjusted to 2.5 with1N HCl and then subjected to dialysis against a 2.5% NaCl solution usingtangential flow filtration on a Pall Minimate™ TFF system. After removalof 5-diafiltration volumes and desalting until the filtrate had aconductivity of <200 S/cm, the retentate solution was collected in alyophilization jar. The solution was frozen in an IPA/dry-ice slurry andlyophilized until dry (3-days). A yield of 1.492 g was obtained as afluffy white solid. The 1H-NMR spectrum (D₂O/DCl) confirmed the expectedstructure.

EXAMPLE 2: SYNTHESIS OF PAAn modified with3-Trifluoromethyl-4-Carboxyphenylboronic acid: PAAn-HCl 50.3% solution(0.5088 g, 2.74 mmol of amine equivalents) was placed in a 30 ml glassvial with a magnetic stir bar and deionized water (9.0 ml). Theresulting clear solution was magnetically stirred, and a pH electrodeintroduced. The pH was adjusted to 8.0 by dropwise addition of a 1N NaOHsolution while stirring. 3-Trifluoromethyl-4-Carboxyphenylboronic acid,98% (0.1148 g, 0.491 mmol) was added to the reaction mixture and theresulting suspension was stirred. After 20 minutes of stirring, the pHhad dropped to 6.8 and some solid remained suspended in solution.Additional NaOH solution was added in portions with stirring to causethe complete dissolution of the suspension. The pH of the resultingclear solution was 7.5. Solid HOBt-hydrate powder (0.0088 g, 0.0514mmol) was then added to the clear reaction solution and dissolved after20 minutes of stirring. Hydrochloric acid (1N) was added to the reactionmixture to lower the pH to 5.4. The reaction solution remained clear.EDC-HCl (0.1143 g, 0.596 mmol) dissolved in 1.0 ml of deionized waterwas then slowly pipetted into the reaction mixture. The clear reactionmixture had a final volume of 10 ml and a pH of 5.6. This reactionmixture was stirred for 18-hours. The reaction mixture was precipitatedinto excess acetone. The precipitated solid was dissolved in water, thepH adjusted to 2.8 with 1N HCl and then subjected to dialysispurification using 6K to 8 k MWCO cellulose membrane dialysis tubing(Spectrum Laboratories) for 8-hours against acidified water (pH=2-3),and then three additional water changes over 2-days. The solutioncontained within the dialysis bag was collected in a lyophilization jar.The solution was frozen in an IPA/dry-ice slurry and lyophilized untildry (3-days). A yield of 263 mg was obtained as a fluffy white solid.The 1H-NMR spectrum (D₂O/DCl) confirmed the expected structure.

EXAMPLE 3: SYNTHESIS OF PAAn modified with3-Fluoro-4-Carboxyphenylboronic acid: PAAn-HCl 50.3% solution (5.964 g,32 mmol of amine equivalents) was placed in a 250 ml beaker with amagnetic stir bar and deionized water (90 ml). The resulting clearsolution was magnetically stirred, and a pH electrode introduced. The pHwas adjusted to 8.0 by dropwise addition of a 1N NaOH solution whilestirring. 3-Fluoro-4-carboxyphenylboronic acid, 97% (1.034 g, 5 mmol)was added to the reaction mixture and the resulting suspension wasstirred. After 20 minutes of stirring, the pH had dropped to 6.8 andsome solid remained suspended in solution. Additional NaOH solution wasadded in portions with stirring to cause the complete dissolution of thesuspension. The pH of the resulting clear solution was 7.5. SolidHOBt-hydrate powder (0.046 g, 0.25 mmol) was then added to the clearreaction solution and dissolved after 20 minutes of stirring.Hydrochloric acid (1N) was added to the reaction mixture to lower the pHto 5.4. The reaction solution remained clear. EDC-HCl (1.152 g, 6 mmol)dissolved in 10 ml of deionized water was then slowly pipetted into thereaction mixture. The clear reaction mixture had a final volume of 120ml and a pH of 5.6. This reaction mixture was stirred for 18-hours. Thereaction mixture was pH adjusted to 2.5 with 1N HCl and then subjectedto dialysis against a 2.5% NaCl solution using tangential flowfiltration on a Pall Minimate™ TFF system. After removal of5-diafiltration volumes and desalting until the filtrate had aconductivity of <200 S/cm, the retentate solution was collected in alyophilization jar. The solution was frozen in an IPA/dry-ice slurry andlyophilized until dry (3-days). A yield of 1.492 g was obtained as afluffy white solid. The 1H-NMR spectrum (D₂O/DCl) confirmed the expectedstructure.

EXAMPLE 4: SYNTHESIS OF PAAn modified with2,3-Difluoro-4-Carboxyphenylboronic acid: PAAn-HCl 50.3% solution(0.5027 g, 2.70 mmol of amine equivalents) was placed in a 30 ml glassvial with a magnetic stir bar and deionized water (9.0 ml). Theresulting clear solution was magnetically stirred, and a pH electrodeintroduced. The pH was adjusted to 8.0 by dropwise addition of a 1N NaOHsolution while stirring. 2,3-Difluoro-4-Carboxyphenylboronic acid(0.1029 g, 0.510 mmol) was added to the reaction mixture and theresulting suspension was stirred. After 20 minutes of stirring, the pHhad dropped to 5.6 and some solid remained suspended in solution.Additional NaOH solution was added in portions with stirring to causethe complete dissolution of the suspension. The pH of the resultingclear solution was 7.5. Solid HOBt powder (0.0082 g, 0.0479 mmol) wasthen added to the clear reaction solution and dissolved after 20 minutesof stirring. Hydrochloric acid (1N) was added to the reaction mixture tolower the pH to 5.5. The reaction solution remained clear. EDC-HCl(0.1018 g, 0.531 mmol) dissolved in 1.0 ml of deionized water was thenslowly pipetted into the reaction mixture. The clear reaction mixturehad a final volume of 10 ml and a pH of 5.5. This reaction mixture wasstirred for 18-hours. The reaction mixture was precipitated into excessacetone. The precipitated solid was dissolved in water, the pH wasadjusted to 2.8 with 1N HCl and the solution was subjected to dialysispurification using 6K to 8 k MWCO cellulose membrane dialysis tubing(Spectrum Laboratories) for 8-hours against acidified water (pH=2-3),and then three additional water changes over 2-days. The contents of thedialysis bag were collected in a lyophilization jar. The solution wasfrozen in an IPA/dry-ice slurry and lyophilized until dry (3-days). Ayield of 235 mg was obtained as a fluffy white solid. The 1H-NMRspectrum (D₂O/DCl) confirmed the expected structure.

EXAMPLE 5: SYNTHESIS OF PAAn modified with3-Dimethylaminomethyl-4-Carboxyphenylboronic Acid: PAAn-HCl 50.3%solution (0.4973 g, 2.67 mmol of amine equivalents) was placed in a 30ml glass vial with a magnetic stir bar and deionized water (9 ml). Theresulting clear solution was magnetically stirred, and a pH electrodeintroduced. The pH was adjusted to 8.0 by dropwise addition of a 1N NaOHsolution while stirring. Solid3-Dimethylaminomethyl-4-Carboxyphenylboronic acid pinacol ester (0.1565g, 0.458 mmol) was added to the reaction mixture and the resultingsuspension was stirred. After 20 minutes of stirring, the pH had droppedto 5.1 and some solid remained suspended in solution. Additional NaOHsolution was added in portions with stirring to cause the completedissolution of the suspension. The pH of the resulting clear solutionwas 8.0. Solid HOBt powder (0.0088 g, 0.0514 mmol) was then added to theclear reaction solution and dissolved after 20 minutes of stirring.Hydrochloric acid (1N) was added to the reaction mixture to lower the pHto 5.5. The reaction solution remained clear. EDC-HCl (0.1048 g, 0.547mmol) dissolved in 1.0 ml of deionized water was then slowly pipettedinto the reaction mixture. The clear reaction mixture had a final volumeof 10 ml and a pH of 5.5. This reaction mixture was stirred for18-hours. The reaction mixture was precipitated into excess acetone, 2×.The collected precipitate was dissolved in de-ionised water and thesolution was frozen in an IPA/dry-ice slurry and lyophilized until dry(3-days). A yield of 271 mg was obtained as a fluffy white solid. The1H-NMR spectrum (D₂O/DCl) confirmed the expected structure, and alsoshowed that the pinacol ester group had been hydrolyzed from the boronicacid.

II. Mucin-Mixing Observational Assay:

The scheme for the mucin mixing assay is shown in FIG. 1.

When a water-soluble mucin glycoprotein is mixed with a solublepolymeric complexing agent, a variety of outcomes may result. Thesolution may remain clear or become cloudy or even opaque. The physicalstate of the mixture may remain homogeneous and flowable, or it maycontain particles or even take on a gel-like appearance. The degree ofopacity and the physical appearance of the mixture provides aqualitative assessment of the interaction between the complexing agentand mucin, and more importantly, the physical properties of the complexthat is formed.

The inventors seek polymers capable of forming extended networkstructures when mixed with mucin. They have found that when certainpolymers are combined with mucin glycoprotein in solution, complexationmay be observed by the appearance of gel-like or opaque mixtures withsubstantial phase separation. Conversely, in the absence ofcomplexation, the solution may remain clear and flowable. In seekingpolymers capable of strong mucin complexation, they are interested inclassifying such mixtures according to well-defined descriptors ofclarity and physical state.

For example, in some cases, a dispersion may be formed. In a dispersion,no particulate is visible. Dispersions may be stable for days withoutsettling or aggregation. In other cases, a suspension of small particlesmay be formed. A suspension may settle out over time (hours, days), butis generally stable and can be re-suspended by mixing. There isliterature precedent for use of a turbidity metric derived from opticalabsorbance at a particular visible light wavelength, and relationship ofthis metric to assess the degree of complexation in some relatedexperiments involving complex formation involving polymers.

However, in some cases, mucin complexation by certain polymers resultsin gross precipitation of larger particles, sometimes with irregular orcomplex morphology. This may be an indication of extended networkformation. In some instances, these precipitated solids have adhesiveproperties, adhering to the walls of the vial in which the mixingexperiment is performed. In some cases, complex fiber-like precipitatesor gels are formed. Furthermore, many cases of gross precipitation areaccompanied by phase separation (syneresis) where the polymer/mucincomplex is deposited as an adherent film or a gel-like mass, and aseparate liquid phase (usually clear or only slightly hazy) is observed.In cases of gross precipitation such as these, it is likely that thedegree of complexation is very high, and it is informative to classifythe morphology and appearance of the mucin/polymer complex in comparisonto related materials.

The objective of this assay is to determine whether the polymers of theinvention are capable of forming an insoluble complex when mixed with asoluble mucin glycoprotein under a set of standard conditions, and toobserve the general properties of the resulting mixture.

The assay outputs are assignment of a clarity descriptor, assignment ofa physical state descriptor and optional descriptions and comments.

Isolation of a water-soluble fraction of Sigma (cat #1778) mucin fromporcine stomach, Type-3 (MPS-3): The protocol is as follows. 1.0 g ofMPS-3 is mixed with 40 ml of MilliQ deionized water in a 50 mL conicaltube. The suspension was left overnight by attaching the tubes to arotating carousel mixer. The 50 ml conical tube was then centrifuged ona Beckman Avanti centrifuge (5,300 rpm for 60 minutes). The supernatantwas poured carefully into a fresh 50 ml conical tube trying to notdisturb the solid pellet at the bottom. Centrifugation was then repeatedon the supernatant (Beckman Avanti centrifuge, 5,300 rpm for 60minutes). The supernatant was again collected into a tared 50 ml conicaltube. The mucin solution was frozen at −80° C. for at least 2 hrs. Thefrozen sample was placed on a lyophilizer for at least 3-days. Thelyophilized product was collected, the tube weighed, and % yieldcalculated, with an expected recovery of 0.60-0.65 g. The solid wasstored at 2-5° C. in the refrigerator.

Preparation of 0.1 M MES-Saline Buffer: 21.3 g of(2-(N-morpholino)ethanesulfonic acid monohydrate) (MES, J. T. BakerBioreagent, >98%) was added into a 1-liter bottle. 9.0 g of SodiumChloride (Fisher Chemical, USP grade) was added into the bottle. A1-liter graduated cylinder was filled with mQ deionized water (DIW, 18MΩ). Approximately 750 ml of DIW was added to the bottle, capped andshaken to dissolve all solids. A magnetic stirring bar and a pHelectrode were added to the bottle, and stirring initiated. 1N of NaOHsolution was added in portions to bring the solution pH to 6.0 (or otherdesired pH), noting the volume of solution added. The required amount ofadditional DIW was added to provide a total volume of 1-liter. Thebottle was capped and the buffer was stored at 2-5° C.

Polymer/mucin mixing turbidity and observational assay: A 1.0% w/wsolution was made of each test polymer using MES-Saline buffer, pHadjusted to 6.0. A 1.0% w/w solution of water-soluble MPS-3 was madeusing MES-Saline buffer, pH adjusted to 6.0. The mucin solution wasslightly hazy. 0.25-0.5 mL of the MPS-3 solution was placed into a2-dram (4 mL) vial. An equivalent volume of the test polymer solutionwas slowly added to the vial. The vial capped and slowly rotated to mixwhile observing any physical changes. One of the following descriptorsfor the clarity of the mixtures can be selected to read the resultingmixture: clear (only slight hazy, similar to initial mucin solution);hazy (more hazy than initial mucin solution, can read text through it);cloudy (non-transparent, can't read through it, but transmits lightwell); opaque (like milk, no transparency, does not transmit much light)and not applicable (material shows gross phase separation). One of thefollowing descriptors on the physical state of the mixtures can beselected to read the resulting mixture: clear; dispersion (no visibleparticles); suspension (very fine particulate is observable),precipitate (large irregular particles, may be adherent) and phaseseparation (films, gels are deposited, a clear or hazy supernatant fluidmay be seen). The mixtures in capped vials were observed for approx. 1hour to note any changes.

Results of mucin-mixing observational assay: Following the protocolabove, a set of test compounds were tested for their ability to forminsoluble complexes with mucin in MES buffer at pH=5.5, 6.0, and 6.5.Polymer solutions were made at least 1-hour before the experiment andallowed to mix gently on a rotisserie. Total dissolution was confirmedfor all test polymer before beginning the experiment.

TABLE 1 Mucin-Mixing Result (clarity/physical-state) Polymer pH = 5.5 pH= 6.0 pH = 6.5 PAAn-HCl 2/B 2/B 2/B Example-1 2/B 2/B 5/E Example-2 2/B5/E 5E Example-3 5/E 5/E 5/E Example-4 5/E 5/E 5/E

The results of the mucin-mixing assay show that when the parent polymer,PAAn-HCl, is mixed with mucin, a hazy dispersion is obtained at all pHvalues. This result suggests that in this case, the polymer/mucincomplex does not form an extended network structure. When the modifiedPAAn of Example-1 is mixed with mucin, a similar hazy dispersion isobserved at pH=5.5 and 6.0. However, at pH=6.5 strong phase separationis observed with a white film depositing on the vessel walls. At thishigher pH value, the polymer of Example-1 is capable of acting as amucin crosslinking agent, forming an extended network structure.

The polymers of Example-2, 3, and 4, are derivatives of the Example-1polymer. They contain an additional electron withdrawing substituentgroup on the phenylboronic acid ring. For these polymers, particularlyExample-3 and 4, the strong phase separation behavior is observed at allpH values examined in this experiment. This additional substitutionactivates the phenylboronic acid resulting in a stronger interactionwith mucin. This stronger interaction causes the observed stronger andmore robust extended network formation at pH=6.0 and in some cases atpH=5.5.

III. Surface Plasmon Resonance (SPR) Study on Mucin-Interacting Polymers

This example describes an investigation of the interaction of solublepolymers with a mucin-coated surface. Surface Plasmon Resonance (SPR)was used to quantify the binding of mucin-interacting polymers insolution flowing over a mucin-coated SPR chip. The binding of thesepolymers was compared to assess how the degree of substitution affectspolymer on binding to the mucin surface.

The SPR experiment was executed using the Bio-Rad XPR36 ProteOn™ SPRinstrument and ProteOn™ LCP sensor chip. Specifically, we observed a setof identical water-soluble poly(allylamine) polymers differing only inthe extent of substitution with the mucophilic group, 3-fluoro-CPBA.Robustness of the surface-deposited material was then challenged byexposure to 1M NaCl. The output of the study is a sensorgram produced bythe Bio-Rad ProteOn™ XPR36 instrument and associated software. Thesensorgram output directly indicates quantitative surface attachment ofpolymer to the modified surface. A fresh, newly purchased LCP sensorchip was used for this experiment.

Preparation of biotinylated Mucin: Biotinylation of MPS-3 water solublefraction: A water soluble fraction MPS-3 was prepared as described underthe mucin-mixing observational assay. Soluble fraction MPS-3 (42 mg) wasdissolved in 30 ml of PBS buffer at pH 7.2 and filtered through a 0.45 mPES syringe filter, then a 0.2 m syringe filter to obtain a clearcolorless solution. (+)-biotin N-hydroxysuccinimide ester (Sigma,#H1759) (25 mg) was dissolved in DMF (2 mL) to obtain a clear solution.The entire 2 mL DMF solution of (+)-biotin N-hydroxysuccinimide esterwas added dropwise via pipette to 18 ml of the MPS-2 PBS solution. Themixture was rocked on a shaker at room temperature for 4-hours coveredin aluminum foil. The reaction mixture was placed in a dialysis tubing(SpectraPor MWCO=6-8 kDa) and the dialysis bag was placed into a5-gallon pail of deionized water with slow stirring. The dialysis wasallowed to continue for at least 48 hours. The retentate (pH found to be6.55) was collected in a lyophilization jar and the material was frozenin a dry-ice/acetone bath and lyophilized. The product was collected asa white fluffy solid (12 mg) and stored in a freezer (−20° C.).

SPR Operations: Coating the ProteOn chip with biotinylated mucin: TheProteOn LCP chip was inserted in to the ProteOn XPR36 instrument. PBSBuffer (pH=6.0) was flown through all 6-lanes of the chip at 100 ul/minfor 3-5 minutes. A solution of biotinylated mucin (5 ug/ml) in PBS(pH=6.0) was flown through all 6-lanes of the chip at 25 ul/min for 600seconds. The sensorgram response was accessed to assure that saturationcoverage is achieved as indicated by stable RU readings. PBS Buffer(pH=6.0) was flown through all 6-lanes of the chip at 100 ul/min for 3-5minutes. The SPR sensorgram response was accessed towards stability ofthe bound mucin layer.

Test polymer-mucin interaction and resistance to wash-off: Six differenttest polymer solutions (4 ug/ml) were flown through the 6-lanes of themucin modified LCP chip at 100 ul/min for 240 seconds. The sensorgramresponse was assessed to assure that saturation coverage is achieved asindicated by stable RU readings. PBS Buffer (pH=6.0) was flown throughall 6-lanes of the chip at 100 ul/min for 3-5 minutes. The SPRsensorgram response was assessed. 1 M NaCl PBS Buffer (pH=6.0) was flownthrough all 6-lanes of the chip at 100 ul/min for 18 seconds. PBS Buffer(pH=6.0) was flown through all 6-lanes of the chip at 100 ul/min for 3-5minutes. The SPR sensorgram response to NaCl treatment was assessed.

Results of SPR study: A stable mucin layer was formed by flowing 5 ug/mlbiotinylated MPS-3 over the Bio-Rad LCP chip: After establishing asteady baseline in flowing buffer solution, exposure of the SPR chip toa solution of 5 ug/ml biotinylated MPS-3 resulted in a steadyaccumulation of material on the LCP chip. The rapid increase in signalintensity rolled-over after 50 seconds and stabilized indicating thatthe surface was saturated with a stable mucin layer.

Binding of PAAn-Fluoro-CPBA polymers to the mucin-coated SPR chip wasaffected by the degree of substitution. After establishing areproducible and stable mucin layer on a new SPR chip, four differentpolymer solutions were simultaneously flowed through the lanes of themicrofluidic chip. The polymer solutions were allowed to flow over thechip for an extended period so that the RU signal had time to roll-overand establish equilibrium for each polymer. All PAAn-fluoro-CPBAderivatives strongly bound to the mucin surface. Surface binding wasfound to be dependent on the level of fluoro-CPBA substitution of thepolymer. PAAn-fluoro-CPBA(15) gave the strongest surface binding (1470RU) followed by PAAn-fluoro-CPBA(10) (1286 RU), and PAAn-fluoro-CPBA(6)(1149 RU). Unsubstituted PAAn also showed strong binding to the mucinsurface, but to a lesser degree (1002 RU).

Exposure of the polymer-bound mucin surface to a strong salt solution(1M NaCl) strongly reduced the adsorbed PAAn surface, but thePAAn-(Fluoro-CPBA) surfaces were stable. After flowing the polymersolutions over the surface long enough to reach a near equilibrium RUsignal, buffer solution again was flowed over the surface demonstratingthe stability of the surface coatings as a stable RU signal in eachlane. Then, a strong salt solution (1M NaCl) was passed over the mucinchip in a short burst (18 seconds) followed by a return to flowingbuffer solution.

The short exposure to NaCl caused the removal of a significant amount ofthe adsorbed PAAn (−26%). In contrast, the RU values for thePAAn-fluoro-CPBA materials showed smaller effects (−2%, −5%, −9%),appearing to be more robust to the strong salt treatment with increasingfluoro-CPBA substitution (see Table 2).

TABLE 2 Conc. Max RU Change in RU lane Polymer (ug/ml) increase* after1M NaCl{circumflex over ( )} 1 Buffer, PBS pH = 6.0 — 0 +2 2 PAAn(NittoBo) 4 1002 −264 (−26%) 3 PAAn-fluoro-CPBA(6) 4 1149 −109 (−9%)  4PAAn-fluoro-CPBA(10) 4 1286 −60 (−5%) 5 PAAn-fluoro-CPBA(15) 4 1470 −35(−2%)

A set of three PAAn-fluoro-CPBA polymers with variable levels ofFluoro-CPBA substitution were evaluated for binding to a mucin modifiedsurface by SPR. The greatest surface binding was seen forPAAn-Fluoro-CPBA(15), with the lower substituted polymers binding to alesser extent. Unsubstituted PAAn also bound the mucin surface, but to asmaller degree.

The SPR surface coated with PAAn-Fluoro-CPBA polymers were stable totreatment with concentrated salt solution. This contrasts the behaviorof unsubstituted PAAn which lost a substantial percentage of boundpolymer when treated with salt. This is likely due to the exclusivelycoulombic association of PAAn with the mucin surface, which iseffectively screened in the presence of a high concentration of salt.For the PAAn-Fluoro-CPBA materials, the combined Coulombic andmucophilic interactions of these modified polymers make their mucincomplex stable to strong salt conditions.

IV. Single Dose Efficacy Study:

Animal Model: The Goto-Kakazaki (GK) rat model was selected based on theextensive literature evidence for response of the GK rat model tosurgical- and device-induced duodenal exclusion. The GK rat model is oneof the most well validated rodent models for Type 2 diabetes drugevaluation. The GK rat is a polygenic non-obese Wistar substrain thatdevelops adult onset Type 2 diabetes early in life. GK rats are an idealType 2 diabetes model, exhibiting characteristics such as retinopathy,nephropathy, neuropathy, and cardiovascular complications similar tothose seen in human disease. Most significantly, this substrain has beenextensively studied in bariatric surgical models, demonstrating animprovement in glucose tolerance subsequent to bariatric surgery similarto that of humans. Oral glucose tolerance testing (OGTT) was used toevaluate the single dose efficacy of the polymer therapy in the GK ratmodel.

General Procedure: Prior to experimentation, all rats were fasted for15-hours with access to water. Animals were split into three test groupswith n=6-7 rats each: (Group A) Vehicle Control which received 0.9%saline; (Group B) Treatment at 40 mg/mL concentration of Example-3dissolved in 0.9% saline; and (Group C) Treatment at 80 mg/mLconcentration of Example-3 dissolved in 0.9% saline. All rats received astandard 1.5 mL volume of their respective solutions delivered by oralgavage in an identical fashion. After 60 minutes, baseline blood glucoselevels were measured. Oral gavage of 40% glucose solution (2.0 g/kg rat)was given immediately after recording the baseline blood glucosemeasurement. Glucose tolerance test samples were then taken from eachrat at 30-, 60-, 90-, and 120-minutes following glucose administration(see FIG. 2). The time dependent blood glucose values were analyzed andplotted using GraphPad Prism 8 software. Two-way ANOVA was applied toraw data to evaluate multiple comparisons between all groups and timepoints. Incremental Area under the Curve was calculated using zero asbaseline for the Y parameter; percentage reduction was calculatedmanually, and t-tests applied to verify significance between treatedgroups versus the vehicle control.

Results: The Treatment groups at 40 mg/mL and 80 mg/mL showed astatistically significant and dose dependent reduction in incrementalArea Under Curve (iAUC) of 31% and 43%, respectively, when compared tothe Vehicle Control (see FIG. 3), thereby demonstrating the efficacy ofthe polymers of the disclosure. Significance was demonstrated at time60, 90, and 120 minutes in each Treatment group with p-values noted inFIG. 2 and FIG. 3 as: * p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

While the invention has been particularly shown and described withreference to specific preferred embodiments, it should be understood bythose skilled in the art that various changes in form and detail may bemade therein without departing from the spirit and scope of theinvention as defined by the appended claims.

1-22. (canceled)
 23. A method for treating a metabolic disorder in asubject, comprising administering to the gastrointestinal tract of asubject in need thereof a therapeutically effective amount of a polymercomprising a repeat unit of Formula (I):

or a pharmaceutically acceptable salt thereof, wherein, R¹, R², and R³are each independently hydrogen, or substituted or unsubstituted alkyl;Y¹ is a direct bond or —NR⁹—; Z is

wherein the —B(OH)₂ is at the 3- or 4-position of the phenyl ring withrespect to Y¹; X¹ and X² are each independently selected from a groupconsisting of hydrogen, halo, and —CF₃, with the proviso that no morethan one of X¹ and X² is hydrogen; R⁹ in each occurrence is hydrogen, orsubstituted or unsubstituted alkyl; n is an integer from 1 to 100,000;and m is an integer from 1 to
 4. 24. The method of claim 23, wherein atleast one of X¹ and X² is fluoro.
 25. The method of claim 23, whereinthe —B(OH)₂ is at the 4-position of the phenyl ring with respect to Y¹.26. The method of claim 23, wherein the repeat unit of Formula (I)comprises the structure:

or a pharmaceutically acceptable salt thereof, wherein m is an integerfrom 1 to 100,000; and X¹ and X² are each independently selected from agroup consisting of hydrogen, halo, and —CF₃; with the proviso that nomore than one of X¹ and X² is hydrogen.
 27. The method of claim 23,wherein the polymer further comprises at least one repeat unit selectedfrom the group consisting of

and pharmaceutically acceptable salts thereof, wherein n is an integerfrom 1 to 100,000.
 28. The method of claim 27, wherein the polymercomprises at least one repeat unit of

or a pharmaceutically acceptable salt thereof, wherein n is an integerfrom 1 to 100,000.
 29. The method of claim 27, wherein the polymercomprises two repeat units, and about 5% to about 50% of the repeatunits comprise Formula (I).
 30. The method of claim 27, wherein thepolymer comprises two repeat units, and about 5% to about 20% of therepeat units comprise Formula (I).
 31. The method of claim 23, whereinthe metabolic disorder is glucose intolerance, type-1 diabetes mellitus(T1DM), type-2 diabetes mellitus (T2DM), prediabetes, hyperlipidemia,obesity, overweight, dyslipidemia, hypertension, hyperglycemia, impairedglucose tolerance, insulin resistance, metabolic syndrome, non-alcoholicsteatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), orpolycystic ovary syndrome (PCOS).
 32. The method of claim 23, whereinthe metabolic disorder is type-2 diabetes mellitus (T2DM).
 33. Themethod of claim 23, wherein administering a therapeutically effectiveamount of the polymer improves glucose homeostasis, reduces insulinresistance, causes weight loss, lowers blood glucose levels, or reducesHbA1C levels in the subject.
 34. The method of claim 23, wherein thepolymer is administered to the subject together with food, or whereinthe polymer is administered to the subject at least 5 minutes and notmore than 12 hours before food.
 35. The method of claim 23, wherein thepolymer is orally administered to the subject.
 36. The method of claim23, wherein the polymer is administered to the subject in a dosage formselected from the group consisting of a powder, a tablet, a caplet, agranule, and a capsule.
 37. The method of claim 23, further comprisingadministering to the subject an additional therapeutic agent.
 38. Themethod of claim 37, wherein the additional therapeutic agent comprises aGLP-1 receptor agonist, a DPP-4 inhibitor, an SGLT-2 inhibitor, aglucosidase inhibitor, an insulin, a biguanide, a sulfonylurea, ameglitinide, a thiazolidinedione, an amylin mimetic, a D2-dopamineagonist, or a bile acid sequestrant.
 39. The method of claim 23, whereinthe method comprises complexing mucin in the gastrointestinal tract ofthe subject.
 40. The method of claim 39, wherein complexing mucin formsa physical barrier between an intestinal lining of the gastrointestinaltract and its luminal contents.
 41. The method of claim 23, wherein thesubject is a human.
 42. The method of claim 23, wherein the subject hasgastroparesis or delayed gastric emptying.
 43. A method for treating ametabolic disorder in a subject, comprising administering to thegastrointestinal tract of a subject in need thereof a therapeuticallyeffective amount of a polymer comprising a structure:

or a pharmaceutically acceptable salt thereof, wherein: X¹ and X² areeach independently selected from a group consisting of hydrogen, halo,and —CF₃, with the proviso that no more than one of X¹ and X² ishydrogen; and each n and m are independently an integer from 1 to100,000, wherein co denotes copolymer.
 44. The method of claim 43,wherein at least one of X¹ and X² is fluoro.
 45. The method of claim 43,wherein the metabolic disorder is glucose intolerance, type-1 diabetesmellitus (T1DM), type-2 diabetes mellitus (T2DM), prediabetes,hyperlipidemia, obesity, overweight, dyslipidemia, hypertension,hyperglycemia, impaired glucose tolerance, insulin resistance, metabolicsyndrome, non-alcoholic steatohepatitis (NASH), non-alcoholic fattyliver disease (NAFLD), or polycystic ovary syndrome (PCOS).
 46. Themethod of claim 43, wherein the polymer is orally administered to thesubject.
 47. A method for treating a metabolic disorder in a subject,comprising administering to the gastrointestinal tract of a subject inneed thereof a therapeutically effective amount of a polymer comprisinga structure selected from the group consisting of:

and pharmaceutically acceptable salts thereof, wherein co denotescopolymer.
 48. The method of claim 47, wherein the metabolic disorder isglucose intolerance, type-1 diabetes mellitus (T1DM), type-2 diabetesmellitus (T2DM), prediabetes, hyperlipidemia, obesity, overweight,dyslipidemia, hypertension, hyperglycemia, impaired glucose tolerance,insulin resistance, metabolic syndrome, non-alcoholic steatohepatitis(NASH), non-alcoholic fatty liver disease (NAFLD), or polycystic ovarysyndrome (PCOS).
 49. The method of claim 47, wherein the polymer isorally administered to the subject.